Polypeptide ligands binding to specific cell surface receptors can activate overlapping but different sets of genes. Recently, a family of latent cytoplasmic transcription factors termed Stats (signal transducer and activator of transcription) have been shown to play important roles in signal transduction pathways activated by interferons (IFNs) and other cytokines. The activities on Stats are dependent on tyrosine phosphorylation which is regulated by Jak (Janus Kinase) protein tyrosine kinases as well as the as-yet-unidentified protein tyrosine phosphatases (PTPases). We have now identified a region of Stat1 which is required for its tyrosine dephosphorylation after IFN stimulation. This region, termed the STD (Stat Tyrosine Dephosphorylation) domain, is highly conserved among all Stat family members isolated so far. Mutations in the Stat STD domain can inhibit the specific tyrosine dephosphorylation of Stat1. In addition, NIH3T3 cells expressing the 'Stat1 STD-deletion mutant protein are hypersensitive to the antiproliferative effect of IFN- gamma. This product will focus on studying the molecular mechanism of the tyrosine dephosphorylation of Stats and the effects of the mutant Stat1 protein on the antiproliferative activity of IFN-gamma. First, we will define and characterize STD domains required for tyrosine dephosphorylation of Stats by site-directed and deletional mutagenesis. Second, we will test our hypothesis that the first and crucial step for the dephosphorylation of Stat1 is through the precise contact of a specific protein tyrosine phosphatase with the Stat1 STD domain. We will determine if the STD domain is sufficient for the recognition of the Stat1 PTPase by competition analysis. We will identify specific proteins) mediating the STD-dependent tyrosine dephosphorylation of Stat1 by in vitro binding analysis and the yeast two-hybrid assays. Third, we will test our hypothesis that the enhanced antiproliferative activity of IFN-gamma mediated by the Stat1 STD-deletion mutant protein is a direct result of altered gene expression induced by this mutant protein. These studies will elucidate the molecular mechanism of Jak-Stat signaling pathways activated by cytokines and growth factors. Results from this research will enhance our ability to design rational therapeutic strategies employing IFNs.